Cell polarity regulates the release of secretory component, the epithelial receptor for polymeric immunoglobulins, from the surface of HT-29 colon carcinoma cells.

The HT-29 human colon carcinoma cell line differentiates in glucose-free medium to an enterocytic phenotype. We previously isolated a series of HT-29 subclones selected for high levels of expression of secretory component (SC), the epithelial receptor for polymeric immunoglobulins. To develop a model system for studying effects of cell polarity on SC expression and release from the cell surface, the HT-29.74 subclone was induced to differentiate in glucose-free medium. Expression of SC was induced by glucose deprivation in both the parental HT-29 cell line and, to an even greater extent, in the HT-29.74 subclone. Prolonged glucose deprivation of HT-29.74 cells resulted in morphological changes consistent with enterocytic differentiation. Metabolic radiolabeling of SC in differentiated HT-29.74 cells indicated that proteolytic cleavage of membrane-bound to free SC occurred both on the cell surface and intracellularly, possibly in a vacuolar apical compartment or intrapeithelial lumen. To study effects of cell polarity on SC release, differentiated HT-29.74 cells were depolarized by culturing in low calcium medium. Within 2 hours after transfer of the cells into low calcium medium, a burst of SC release was observed concomitant with cell depolarization. Subsequently, release of SC declined significantly and remained low as long as cells were maintained in a depolarized state. The extent of cell depolarization could be controlled by varying the extracellular calcium concentration or by substituting the divalent cation Sr++, which partially prevents depolarization, for Ca++. In either case, the magnitude of the initial burst and subsequent decline in release of SC was proportional to the extent of cell depolarization. We conclude that cell polarity plays an important role in controlling the release of SC in intestinal epithelial cells, most likely by regulating the distribution of membrane-bound SC and SC protease, which are on the basolateral and apical cell surfaces, respectively, in differentiated cells.     

Affinity-purified site-directed antibody recognizes the entire annexin protein family

Annexins are a family of calcium/phospholipid binding proteins sharing strong sequence similarities. A site-directed affinity-purified antibody was produced against the consensus peptide, K A M K G L G T D E. This antibody recognizes all six annexin proteins in the purified state and in total-protein tissue extracts. The antibody should prove useful in identifying functional domains of this protein family.     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Co-distribution of annexin VI and actin in secretory ameloblasts and odontoblasts of rat incisor

Summary Annexin VI and actin were detected by immunoblot analysis in the enamel- and dentin-related portions of dental tissues. Annexin VI was found mainly in the particulate fraction whereas actin was detected in both the soluble and particulate fractions. By immunoelectron microscopy, annexin VI antibodies conjugated with colloidal gold were seen to label the mitochondria, the cytosol and the nucleus of secretory ameloblasts and odontoblasts of rat incisor. In the processes of these cell, the plasmalemmal undercoat was labeled. Antiactin antibodies labeled the desmosome-like junctions, the cytosol, and the mitochondria of the cell bodies. Extensive labeling was seen at the periphery of the Tomes' processes and odontoblast processes. These results suggest that annexin VI may play a role in Ca²⁺-regulation in the cell bodies, especially as a calcium receptor protein in the mitochondria. Moreover, annexin VI and actin seem to be co-distributed in secretory processes. Thus, these proteins might be both involved in exocytotic and endocytotic events.     

Site-directed mutagenesis of the human chorionic gonadotropin beta-subunit: bioactivity of a heterologous hormone, bovine alpha-human des-(122-145)beta

Human CG contains an alpha-subunit, common to the pituitary glycoprotein hormones, and a hormone-specific beta-subunit, but unlike the pituitary beta-subunits, hCG beta is characterized by an O-glycosylated carboxy-terminal extension. A mutant beta-subunit, des-(122-145)hCG beta, was prepared using site-directed mutagenesis, and the pRSV expression plasmids were transfected into Chinese hamster ovary cells that produce the bovine alpha-subunit (b alpha). The mutant beta-subunit binds to b alpha, and the heterologous gonadotropin, b alpha-des-(122-145)hCG beta, was capable of stimulating steroidogenesis in cultured Leydig tumor cells (MA-10) to the same extent as standard hCG. When compared with the heterologous gonadotropin, b alpha-hCG beta wild type, the hybrid hormone with the truncated hCG beta exhibited equal potency, within the accuracy of the RIAs used to determine hormone concentrations, and gave a similar time course of steroidogenesis. Interestingly, these transformed Leydig cells do not distinguish between the steroidogenic potencies (as measured by progesterone production) of hCG and human LH (hLH) as do some preparations of normal rodent Leydig cells (as measured by testosterone production). However, the MA-10 cells were able to distinguish hCG from hLH based on their cAMP response; the latter produced a greater response at both maximal and submaximal gonadotropin concentrations. The two expressed heterologous gonadotropins were equipotent in their abilities to stimulate cAMP and gave similar time courses of cAMP accumulation in MA-10 cells. Thus, the carboxy-terminal extension of hCG beta is not required for association with the alpha-subunit nor for functional receptor binding, as judged by cAMP accumulation and progesterone production in MA-10 cells.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Evidence for "twisted plane" undulations in golden hamster sperm tails

Motile spermatozoa from the golden hamster have been arrested by rapid freezing and then fixed with glutaraldehyde at low temperature after substitution with ethylene glycol. As far as can be judged, the flagellar waveforms thus stabilized are similar to those seen in living sperm; in contrast, fixation in glutaraldehyde, without prior freezing, induces agonal changes in flagellar conformation. The characteristics waveform after freeze substitution contains three bends. Approx. half of these flagella are entirely planar. The rest are three dimensional, with the third bend displaced in a regular way from the plane containing the proximal two bends. From the geometry of these flagella, it is concluded that the plane of action of a given bending cycle undergoes a clockwise twist (from a forward viewpoint) as the cycle is succeeded by new bending cycles. This "twisted plane" undulation is quite different from helical movement. The twisting seems to occur abruptly, between cycles, as if each bending cycle has a preferred plane of action. The mechanism underlying the twisting is uncertain. However, on the basis of the angular displacements between the preferred planes, and the findings from electron microscopy, the following idea is presented as a working hypothesis: that, if the most proximal plane of bending is topographically determined by peripheral doublet 1, then successive distal planes of action are influenced predominantly by doublets 2, 3, etc., in clockwise sequence. The merits and weaknesses of this hypothesis are discussed.     

Impact of human activities on the reproduction of Hooded Vultures Necrosyrtes monachus in Burkina Faso

During the last decades, the critically endangered Hooded Vulture Necrosyrtes monachus has strongly declined across its African range. Although direct persecution has been suggested as a major cause of this decline, little is known about the impact of humans on reproductive output in West Africa. We studied the impact of human activities on the reproductive output of Hooded Vultures in the Garango area of Burkina Faso. Twenty and 56 nesting attempts were monitored, respectively, during the breeding season in 2013/14 and 2014/15, to determine reproductive success and identify causes of nest failure. Annual breeding success varied between 0.68 and 0.71 chicks fledged per breeding pair per year and productivity was assessed at 0.57 chicks fledged per territorial pair in 2014/15. The main threats imposed by humans were poaching of eggs, chicks and collection of nest materials, leading to 20% (13 out of 64 breeding attempts) of nest failures over the two years. An additional important reason for nest failure was the pruning and (partial) cutting of nest trees. Despite this high level of human interference, we found that Hooded Vulture nest success increased with proximity to human settlements, probably because breeding vultures benefit from protection by people against persecution and disturbance.